The invention relates to lanthanide chelates, their manufacture and characterization and their use in bioanalysis, preferably in fluorescence spectroscopy.
Using lanthanide complexes in fluorescence spectroscopy is already known. U.S. Pat. No. 4,374,120 describes Eu and Tb chelates as fluorescent markers with a relatively long fluorescent time of 50 to 1000 microseconds, ligands are aminopolycarboxylic acids, among other things. In addition, it is also known that some lanthanide fluorescence chelate complexes are particularly suited for time-resolved fluorimetry, wherein TB (III)-BPTA—NHS and Eu(III) estrogen are preferred and the former is used in a DNA hybridization assay (K. Matsumoto et al., RIKEN Review 35, May 2001). Lanthanide chelates are used according to WO 00/01663 in HTRF (homogenous time-resolved fluorescence) assays. Using cyanine and indocyanine dyes in biomedicine is known (U.S. Pat. No. 6,217,848, U.S. Pat. No. 6,190,641 with additional evidence). DE 42 22 255 describes marking reagents with a lanthanide ion chelating structure for use in gene probe diagnostics. Preferred as a lanthanide ion chelating structure are pyridine derivates, spacers are polyalkyl amine and polyethylene glycol, and furocumarin derivates are photosensitive. In an article in the Journal of Alloys and Compounds 1995, 225, 511-14 on Page 112, H. Takalo et al. describe Tb(III) and Eu(III) chelates and their luminescence yield. To determine phosphorylation activities, cryptates are used in DE 698 13 850, which contain a rare earth molecule such as TB, Eu, Sm, Dy, Nd, a complexing agent like bispyridine and which are used as the fluorescent donor bond. I. Hemmilää and S. Webb describe principles of time-resolved fluorimetry TRF with lanthanide chelates for drug screening in DDT, 1997, 2, 373-381. A sensor for detecting nucleic acids that uses rare earth dyes as fluorophores in a preferred embodiment is described in DE 102 59 677.
For use in bioanalysis by measuring the energy transfer of a donor to an acceptor, it is necessary to have compounds available that are capable of this transfer of energy. One possibility is fluorescence resonance energy transfer (FRET) as a special form of energy transfer, which is based on an interaction of two spatially separated dipoles, one of which (donor) is electronically excited. If both dipoles are in resonance with one another, the excitation energy of the fluorescent donor can be transferred nonradiatively to an acceptor. Because of its high sensitivity and the strong dependence on the distance between the donor and acceptor, FRET has found wide-ranging application in the identification and characterization of biologically relevant substrates. A current development concerns the use of FRET systems in homogenous fluorescent assays (sequence marking, fold boundary marking (proteins), DNS). In the process, the antigens and antibodies bought into reaction are marked with a fluorophore group, in which the one fluorophore functions as an energy donor and the other as a corresponding energy acceptor.
A comprehensive application of the FRET principle for the direct detection of molecule-molecule interactions in clinical laboratory diagnostics as well as in combinatorial pharmasynthesis requires the availability of these type of donors and acceptors, which are identified by efficient spectroscopic absorption and emission behavior in the long-wave spectral range, wherein the excitation wavelengths of the donor must be >350 nm in order to avoid an excitation of the biological substrate. Another essential criterion for their applicability in biological test methods is the formation of a stable, covalent biopolymer fluorophore compound without the stability and the biological activity of the marked molecule being negatively impacted in the process.
In view of the need for knowledge in this field, appropriate donor-acceptor systems are being sought, which meet the requirements for precision and short-term data availability, because known donor-acceptor systems have several disadvantages. Thus the transfer of energy from the donor to an acceptor is frequently not satisfactory and the sensitivity of the analytic method as a whole also suffers from this. Moreover, the complex stability of the donor frequently does not suffice and its water solubility is insufficient.
The causes of the described disadvantages lie to begin with in the composition, i.e., in the structure of the donor compounds. Creating compounds with high stability of the donor compound with the required spectroscopic properties of absorption and emission has not been successful so far. In addition, the acceptor dyes that have been used thus far are not the compounds of choice, because they are not coordinated in terms of their absorption maximum with respect to the emission spectrum of the donor and it has not been possible yet to guarantee sufficient solubility of the acceptor dye.
For a targeted application of the fluorophores, the priority development claim lies with the synthesis of new compounds in the increase of efficiency of the energy transfer from the donor to the corresponding acceptor and thus in an increase in the sensitivity of the analytical method. This claim can be taken into consideration by use of a lanthanide complex as an energy donor, in which a complexon with a special chromophorous group related to the application chelates the lanthanide(III)ion. The complex formation of the actual fluorophore, the lanthanide(III)ion, by a complexon should guarantee a high level of complex stability to the donor used and good solubility in an aqueous medium. The characteristic feature of these lanthanide(III) complexes used for the first time lies in the modification of the ligand system by substituents (designated as antenna in the following), which absorb in the planned wavelength range and can be coordinated with the excitation wavelength as a result.
The use of lanthanide(III) compounds and energy donors in fluorescence analysis should offer spectroscopic advantages as compared with organic donors with respect to sensitivity and signal-to-noise ratio and must therefore open up a multitude of application aspects, in particular also in combinatorial pharmasynthesis. The reason for this is the spectroscopic properties that are characteristic for the lanthanide(III) complexes, such as the large STOKES displacement, the line-like emission connected with high intensity as well as long lifetimes of the excited states. In addition, besides a time-resolved spectrogram, a decisive advantage of lanthanide(III) compounds lies in the favorable donor-acceptor distances, which make better signal separation and signal intensification possible vis-à-vis an incompletely marked substrate and permit a generally large label.